Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation.

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.

Structures and Surface Properties of "Cyclic" Polyoxyethylene Alkyl Ethers: Unusual Behavior of Cyclic Surfactants in Water.

The cyclization of amphiphiles has emerged as an attractive strategy for inducing remarkable properties in these materials without changing their chemical composition. In this study, we successfully synthesized three cyclic polyoxyethylene dodecyl ethers (c-POEC12's) with different ring sizes and explored the effects of their topology on their surface and self-assembly properties related to their function, comparing them with those of their linear counterparts (l-POEC12's). The surface activity of the c-POEC12's remained almost constant despite the change in their hydrophobic and hydrophilic balance (HLB) value, while that of the l-POEC12's decreased with an increase in the HLB value as general surfactants. In contrast to the normal micelles seen in the case of the l-POEC12's (3.4-9.7 nm), the cyclization of the POEC12's resulted in the formation of large spherical structures 72.8-256.8 nm in size. It also led to a dramatic decrease of 28 °C in the cloud point temperature. Furthermore, the cyclization of the POEC12's markedly suppressed the rate of protease hydrolysis caused by the surfactants. The initial rate of reduction of a detergent enzyme from Bacillus licheniformis was increased by more than 40% in the case of c-POE600C12 and c-POE1000C12, even though they exhibited surface activities almost equal to or higher than those of their linear counterparts. These results suggest that cyclization induces unusual aqueous behaviors in POEC12, making the surfactant milder with respect to detergent enzymes while ensuring it exhibits increased surface activity.

Surfactant-like properties of an amphiphilic α-helical peptide leading to lipid nanodisc formation.

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that wrap themselves around the circumference of a lipid bilayer in a beltlike manner. In this study, an amphiphilic helical peptide that mimics helix 10 of human apoA-I was newly synthesized by solid phase peptide synthesis using Fmoc chemistry, and its physicochemical properties, including surface tension, self-association, and solubilization abilities, were evaluated and related directly to nanodisc formation. The synthesized peptide having hydrophobic and hydrophilic faces behaves like a general surfactant, affording a critical association concentration (CAC) of 2.7 × 10(-5) M and a γCAC of 51.2 mN m(-1) in aqueous solution. Interestingly, only a peptide solution above its CAC was able to microsolubilize L-α-dimyristoylphosphatidylcholine (DMPC) vesicles, and lipid nanodiscs with an average diameter of 9.5 ± 2.7 nm were observed by dynamic light scattering and negative stain transmission electron microscopy. Moreover, the ζ potentials of the lipid nanodiscs were measured for the first time as a function of pH, and the values changed from positive (20 mV) to negative (-30 mV). In particular, nanodisc solutions at acidic pH 4 (20 mV) or basic pH 9 (-20 mV) were found to be stable for more than 6 months as a result of the electrostatic repulsion between the particles.

Encapsulation of ion pairs in extended, self-assembled structures.

Encapsulation of ion pairs in small spaces that are isolated from the medium is expected to result in amplified interactions between the ions. Yet, sequestration of ion pairs in self-assembled capsules is complicated by competition of the acids and bases for binding directly to the assembly components. We describe here a hydrogen-bonded capsule 1.2(8).1 that accommodates two γ-picolines and two acids as ion pairs. The supramolecular structure of the discrete 14-component assembly is characterized by NMR spectroscopy. The structure reveals the acids in the tapered ends of the capsule and γ-picoliniums near the glycoluril spacers in the capsule's center. Similar acid-base ion pairs are also obtained with 4-ethylpyridine, γ-picoline with difluoroacetic acid, and γ-picoline with trifluoromethanesulfonic acid. The (1)H NMR spectrum of the γ-picoline/trifluoroacetic acid ion pair shows a signal at δ = 18.7 ppm, indicating the acidic proton is in contact with both the picoline nitrogen and the trifluoroacetate oxygen. Further details about the unusual structures of ion pairs in small spaces are reported.

Protein recognition by a self-assembled deep cavitand monolayer on a gold substrate.

This paper details the first use of a self-folding deep cavitand on a gold surface. A sulfide-footed deep, self-folding cavitand has been synthesized, and its attachment to a cleaned gold surface studied by electrochemical and SPR methods. Complete monolayer formation is possible if the cavitand folding is templated by noncovalent binding of choline or by addition of space-filling thiols to cover any gaps in the cavitand adsorption layer. The cavitand is capable of binding trimethylammonium-tagged guests from an aqueous medium and can be deposited in 2 × 2 microarrays on the surface for characterization by SPR imaging techniques. When biotin-labeled guests are used, the cavitand:guest construct can recognize and immobilize streptavidin proteins from aqueous solution, acting as an effective supramolecular biosensor for monitoring protein recognition.

Metallosurfactants Consisting of Amphiphilic Ligands and Transition Metals: Structure, Bonding, Reactivity, and Self-assembling Property.

Metallosurfactants are emerging as a relatively new class of surfactants whose ligand moieties bind to various transition metals. Because transition metal centers are incorporated into the surfactant frameworks, they can form various self-assembled structures with metallic interfaces such as micelles, vesicles, and lyotropic liquid crystals. To reduce the lability of transition metal complexes under aqueous conditions, various amphiphilic ligands have been developed as surfactant frameworks. This review discusses some aspects of the design and chemical structures of amphiphilic ligands, as well as focus on various functions and types of chemical bonds present in metallosurfactants.

Enrichment and Isolation of Surfactin-degrading Bacteria.

A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited microbial growth as well as surfactin degradation, were selected and further investigated. After several successive cultivations, nanopore sequencing of full-length 16S rRNA genes with MinIONTM was used to analyze the bacterial species in the enrichment cultures. Variovorax spp., Caulobacter spp., Sphingopyxis spp., and Pseudomonas spp. were found to be dominant in these surfactin-degrading mixed cultures. Finally, one strain of Pseudomonas putida was isolated as a surfactin-degrading bacterium. This strain degraded 1 g/L surfactin below a detectable level within 14 days, and C13 surfactin was degraded faster than C15 surfactin.

Au(i)-, Ag(i)-, and Pd(ii)-coordination-driven diverse self-assembly of an N-heterocyclic carbene-based amphiphile.

Au(i)-, Ag(i)-, and Pd(ii)-coordination-driven diverse self-assembly of an N-heterocyclic carbene (NHC)-based amphiphile was demonstrated herein. The transition metals had significant effects over the whole system, setting the self-assembly direction of the NHC-based amphiphile. More specifically, Au(i)- and Ag(i)-coordination to the NHC-based amphiphile promoted the formation of spherical and hexagonal structures, while Pd(ii)-coordination promoted the formation of cylindrical and lamellar structures.

Hydrogels composed of organic amphiphiles and alpha-cyclodextrin: supramolecular networks of their pseudorotaxanes in aqueous media.

Mixtures of N-alkyl pyridinium compounds [py-N-(CH(2))(n)OC(6)H(3)-3,5-(OMe)(2)](+)(X(-)) (1bCl: n = 10, X = Cl; 1cBr: n = 12, X = Br) and alpha-cyclodextrin (alpha-CD) form supramolecular hydrogels in aqueous media. The concentrations of the two components influences the sol-gel transition temperature, which ranges from 7 to 67 degrees C. Washing the hydrogel with acetone or evaporation of water left the xerogel, and (13)C CP/MAS NMR measurements, powder X-ray diffraction (XRD), and scanning electron microscopy (SEM) revealed that the xerogel of 1bCl (or 1cBr) and alpha-CD was composed of pseudorotaxanes with high crystallinity. (13)C{(1)H} and (1)H NMR spectra of the gel revealed the detailed composition of the components. The gel from 1bCl and alpha-CD contains the corresponding [2]- and [3]pseudorotaxanes, [1b x (alpha-CD)]Br and [1b x (alpha-CD)(2)]Br, while that from 1cBr and alpha-CD consists mainly of [3]pseudorotaxane [1c x (alpha-CD)(2)]Br. 2D ROESY (1)H NMR measurements suggested intermolecular contact of 3,5-dimethoxyphenyl and pyridyl end groups of the axle component. The presence of the [3]pseudorotaxane is indispensable for gel formation. Thus, intermolecular interaction between the end groups of the axle component and that between alpha-CDs of the [3]pseudorotaxane contribute to formation of the network. The supramolecular gels were transformed into sols by adding denaturing agents such as urea, C(6)H(3)-1,3,5-(OH)(3), and [py-N-nBu](+)(Cl(-)).

Synthesis of a Bolaamphiphilic Alkenyl Phosphonic Acid by Ru-catalyzed Olefin Cross Metathesis Reaction.

We report the synthesis of bolaamphiphilic alkenyl phosphonic acid (BPC12) through the olefin crossmetathesis reaction of vinylphosphonic acid with 1,11-dodecadiene in the presence of a Ru-carbene catalyst. BPC12 possesses two trans-P-C=C moieties and is thus readily soluble in water up to 3.4 g L-1, as confirmed by 1H nuclear magnetic resonance (NMR) measurements. Surface tension measurements revealed that BPC12 reduced the surface tension of water from 72.0 to 47.0 mN m‒1. The occupied area per molecule at the air/water interface (A) of BPC12 (216 Å2) was ten times larger than that of dodecenyl phosphonic acid PC12 (23 Å2). Moreover, dynamic light scattering measurement of an aqueous BPC12 solution (5 mM) revealed the formation of large aggregates with an average diameter of 81.8±27.0 nm.

Synthesis of an N-Heterocyclic Carbene-based Au(I) Coordinate Surfactant: Application for Alkyne Hydration Based on Au Nanoparticle Formation.

In this study, an N-heterocyclic carbene (NHC)-based metal coordinate surfactant (MCS), NHC-Au-MCS, in which the NHC framework afforded the bonding of the Au(I) at the linkage of the hydrophilic and hydrophobic moieties, was synthesized. The structure of NHC-Au-MCS was confirmed by 1H and 13C NMR spectroscopic measurements together with elemental analysis. Matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization (LDI), and electrospray ionization mass spectrometry (ESI-MS) indicated the distinct reactivity of NHC-Au-MCS, such as the exchange of Br to Cl and the formation of a cationic Au complex, where the two NHC ligands were coordinated to an Au(I) center upon laser activation. The surface tension and dynamic light scattering (DLS) measurements revealed that the coordination of Au(I) to NHC reduced the critical micelle concentration (CMC) of NHC-Au-MCS (1.3×10-5 M), which resulted in the formation of micelles at concentrations higher than the CMC in water. We also confirmed that the surface-active Au(I) complex of NHC-Au-MCS catalyzed the hydration of 1-dodecyne to 2-dodecanone in water in the absence of an organic solvent. On the basis of the detailed mechanistic investigations regarding the reactivity of NHC-Au-MCS, we revealed that NHC-Au-MCS partially translated into Au nanoparticles (AuNPs), which facilitated alkyne hydration. These mechanistic studies were supported by UV-vis measurements, transmission electron microscopy (TEM), and LDI-MS.

Evaluation of Yield and Surface Tension-lowering Activity of Iturin A Produced by Bacillus subtilis RB14.

Bacillus subtilis RB14 produces the lipopeptide antibiotic iturin A by submerged and biofilm fermentation. In this study, we optimized the conditions for iturin A production in a jar fermentor. The maximum yield of iturin A was 932 mg L-1 after 120 h. The surface tension of water decreased from 72.0 to 39.0 mN m-1 as the concentrations of C14 iturin A increased, indicating that C14 iturin A behaves as a surfactant in water. The critical micellar concentration obtained from the intersection of two fitted lines was 1.2 × 10-4 M. Moreover, the surface tension of water decreased as the length of the alkyl chain of iturin A increased.

Spontaneous Vesicle Formation of Monododecenyl Phosphonic Acid in Water.

We report the synthesis of amphiphilic dodecenyl phosphonic acid PC12 from vinylphosphonic acid, a reactive phosphonic acid intermediate. The trans-P-C=C moiety enabled PC12 to disperse well in water. Surface tension and dynamic light scattering measurements revealed that PC12 exhibited high surface activity and reduced the surface tension of water from 72.0 to 23.6 mN/m, thereby resulting in the spontaneous formation of aggregates even in a dilute aqueous solution (critical aggregation concentration (CAC) = 4.8 × 10-4 M). In contrast to modern lipids with double-tailed structures, the PC12 of simple singletailed structure spontaneously formed bilayered vesicles, without an external energy supply. Compared with the strength of hydrogen bonds formed by the long, saturated alkyl chain of dodecyl phosphonic acid (DPA), the strength of PC12 intermolecular hydrogen bonds was weaker. The melting point of PC12 was approximately 20°C lower than that of DPA. These results indicate that the trans-P-C=C moiety was considerably important for spontaneous vesicle formation in water. Preliminary modeling of the morphological transitions of the closed bilayer structures in the vesicles was then conducted, by varying the pH and adding an α-helical peptide scaffold.

Surface Activity and Ca2+-Dependent Aggregation Property of Lichenysin Produced by Bacillus licheniformis NBRC 104464.

Bacillus licheniformis NBRC 104464 produces a cyclic lipopeptide different from surfactin. After we performed liquid chromatography fractionation and purification, we used structural analyses to identify the cyclic lipopeptide as lichenysin. Surface tension measurements of lichenysin sodium salt in water yielded a critical micelle concentration (CMC) of 1.0×10-5 M. The surface tension at the CMC was 28.9 mN/m. Comparative analysis of Ca2+-influenced micellar aggregation of lichenysin and surfactin revealed that the formation rate of the lichenysin-Ca2+ complex aggregate remained low up to a [Ca2+]/[lichenysin] molar ratio of 80, whereas the surfactin-Ca2+ complex formed micellar aggregates at the same molar ratio. Further excessive addition of Ca2+ to the micellar solution of lichenysin induced higher turbidity than surfactin.

Lipid Nanodisc Formation using Pxt-5 Peptide Isolated from Amphibian (Xenopus tropicalis) Skin, and its Altered Form, Modify-Pxt-5.

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that accumulate around the circumference of a lipid bilayer. In this study, Pxt-5, which is an antimicrobial peptide isolated from the skin of Xenopus tropicalis, and its modified peptide (Modify-Pxt-5) were synthesized by solid-phase peptide synthesis (SPPS).Their surface properties, which are an important factor in inducing nanodisc formation, were investigated by circular dichroism (CD) spectroscopy, surface tension measurement, phospholipid vesicle clearance assay, and negative-staining transmission electron microscopy (NS-TEM). The α-helicity of Pxt-5 (8.4%) improved drastically to 45.6% by four amino-acid substitutions (Modify-Pxt-5). Both the peptides, having hydrophobic and hydrophilic faces, behaved like general surfactants, and the surface activity of Modify-Pxt-5 (CAC: 9.5×10-5 M, γCAC: 30.3 mN·m-1) was much higher than that of Pxt-5 (CAC: 7.9×10-5 M, γCAC: 38.1 mN·m-1). A turbid L-α-dimyristoylphosphatidylcholine (DMPC) vesicle solution (T = 0.3%) quickly turned transparent upon addition of Pxt-5 or Modify-Pxt-5. After twelve hours, the transmittance of vesicle solution with Modify-Pxt-5 (T = 96.2%) was found to be higher than that of vesicle solution with Pxt-5 (T = 83.5%), and then the micro-solubilized solutions were observed by NS-TEM. Interestingly, nanodisc structures were found in the vicinity of DMPC vesicles in both the images, and the average diameter of the nanodiscs was 11.2 ± 6.0 nm for those containing Pxt-5 and 10.8 ± 5.8 nm for those containing Modify-Pxt-5. It was also found that Modify-Pxt-5 effectively self-assembles into nanodiscs compared to Pxt-5 without any substitutions.

Self-assembling Properties of an N-Heterocyclic Carbene-based Metallosurfactant: Pd-Coordination Induced Formation of Reactive Interfaces in Water.

In this study, an N-heterocyclic carbene (NHC)-based metallosurfactant (MS), NHC-PdMS, was synthesized, where Pd(II) was bound to the NHC framework via a robust Pd-carbene bond with NEt3 as a co-ligand. Surface tension measurements revealed that the critical micelle concentration (CMC) of NHC-PdMS (1.8×10-4 M) was one order of magnitude lower than that of its MS precursor (imidazolium bromide). Coordination of the MS precursor and NEt3 to Pd(II) also influenced micelle size; the hydrodynamic diameters of NHC-PdMS and the MS precursor were observed to be 25.8±5.6 nm and 2.5±0.3 nm, respectively. Furthermore, small angle X-ray scattering measurements indicated that NHC-PdMS exhibited liquid crystalline behavior above 26 wt%, with a spacing ratio of 1:2:3 for the first, second, and third Bragg peaks. To understand the role of the reactive interface, NHC-PdMS was also applied to aqueous catalytic reactions. Owing to its low CMC value, a catalytic amount of NHC-PdMS (3 mol%) provided the reactive interface, which facilitated the aqueous Mizoroki-Heck reaction of various aryl iodides and styrene in good yields (72-95%). These results suggest that MS formation results in a drastic change in selfassembling properties, which are important for the development of highly reactive chemical interfaces in water.

Competing supramolecular assembly of amphiphiles to form micelles or pseudorotaxanes.

[reaction: see text] Micelles of an amphiphile that encapsulate the added dye in water can be released, upon addition of alpha-CD, to form pseudorotaxane. The equilibrium between the micelles and the pseudorotaxanes and the absorption spectra of solution are controlled by temperature reversibly.

Screening of a Bacillus subtilis Strain Producing Multiple Types of Cyclic Lipopeptides and Evaluation of Their Surface-tension-lowering Activities.

Sixty Bacillus subtilis strains were investigated for their ability to produce cyclic lipopeptides (CLPs). Among them, B. subtilis NBRC 109107 produced at least three types of CLPs by high-performance liquid chromatography (HPLC) analysis, and these CLPs were thought to be surfactin, iturin A, and fengycin by polymerase chain reaction amplification of respective CLP synthetase-encoding genes. However, after HPLC fractionation and purification, structural analysis of the CLPs revealed that these were surfactin homologues, iturin A, and unknown CLPs, whose surface-tension-lowering activities were 29.4, 56.7, and 48.6 mN/m, respectively. By contrast, fengycin was not detected.

pH-induced conformational change of natural cyclic lipopeptide surfactin and the effect on protease activity.

The cyclic lipopeptide surfactin (SF) is one of the promising environmental friendly biosurfactants abundantly produced by microorganisms such as Bacillus subtilis. SF shows excellent surface properties at various pH, together with lower toxicity and higher biodegradability than commonly used petroleum-based surfactants. However, the effect of the dissociation degree of SF on self-assembly is still incompletely understood, even though two acidic amino acid residues (Asp and Glu) are known to influence eventual surface and biological functions. Here, we report changes in the secondary structure of SF induced by increased pH, and the effect on protease activity. We found that the ß-sheet and ß-turn formation of SF are significantly enhanced through increased dissociation of Asp and Glu as revealed by a titration experiment of SF solution to estimate apparent pK1 and pK2 values together with circular dichroism spectroscopy. We also studied the activity of the common detergent enzyme subtilisin in SF solution at above its pK2 (pH 7.6) to understand the role of the dissociation degree in the interaction with the protein. The mixing of SF having a unique cyclic topological feature with subtilisin suppressed the decrease in protease activity observed in the presence of synthetic surfactants such as sodium dodecyl sulfate and polyoxyethylene alkyl ether. Thus, SF has great potential for use in laundry detergent formulations, to improve the stability and reliability of detergent enzymes.

Interfacial and emulsifying properties of soybean peptides with different degrees of hydrolysis.

In this study, the effects of the degree of hydrolysis on the interfacial and emulsifying properties of soybean peptides were evaluated based on surface and interfacial tension, dynamic light scattering (DLS), and freeze-fracture transmission electron microscopy (FF-TEM) analyses. Of the five evaluated soybean peptides (SP95, SP87, SP75, SP49, and SP23), those with higher degrees of hydrolysis (SP95 and SP87) did not exhibit noticeable surface-active properties in water, whereas those with relatively low degrees of hydrolysis (SP75, SP49, and SP23) exhibited remarkable surface tension-lowering activity. The latter set (SP75, SP49, and SP23) also formed giant associates with average sizes ranging from 64.5 nm to 82.6 nm above their critical association concentration (CAC). Moreover, SP23 with the lowest degree of hydrolysis exhibited excellent emulsifying activity for soybean oil, and FF-TEM analysis demonstrated that the emulsions were stabilized by a lamella-like multilayer peptide structure on the oil droplets that prevented coagulation. The peptide with the lowest degree of hydrolysis (SP23) was effective not only for soybean oil emulsification, but also for the emulsification of liquid paraffin and silicon oil that are generally difficult to emulsify.